Yarrowia lipolytica engineering bacterium for producing isopentene aromatic compound as well as construction method and application of Yarrowia lipolytica engineering bacterium

Abstract

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to yarrowia lipolytica engineering bacteria for producing isopentene aromatic compounds as well as a construction method and application of the yarrowia lipolytica engineering bacteria. Specifically, synthesis of two precursors of p-coumaric acid and dimethyl aryl pyrophosphate is engineered in parallel in two haploid yarrowia lipolytica strains. And then mating the receptor strain with the donor strain to construct the diploid capable of efficiently synthesizing the drpanin and the artepillin C. The invention further discloses a preparation method of the diploid of the drpanin and the artepillin C. By using a similar method, a strain capable of producing the 8-prenylnaringenin is also constructed. According to the present invention, the basis is provided for the sustainable production of drpanin, artepillin C and 8-prenylnaringenin, and the new way is provided for the rapid engineering of the yeast to produce other isopentenylated compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and fermentation engineering, and specifically relates to an engineering bacterium of Yarrowia lipolytica producing isopentenylated aromatic compounds, a construction method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The prenylation of aromatic compounds is an important step in the biosynthesis of many biologically active compounds. To date, about 1,000 prenylated aromatic compounds have been characterized, many of which have antioxidant, antiviral, and antitumor activities. For example, the prenylation of p-coumaric acid produces drupanin and artepillin C. Drupanin and artepillin C have been identified as key bioactive compounds in Brazilian green propolis. In addition, prenylated flavonoids are a subclass of plant prenylated aromatic compounds. 8-Prenylnaringenin is present in hops and has been reported to have a variety of biological activities. However, the content of prenylated aromatic compounds in nature is usually very low, which limits their development and utilization.

[0004] The unconventional yeast Yarrowia lipolytica is a generally considered safe microorganism that has been widely used to synthesize natural products. However, its application in the synthesis and production of prenylated aromatic compounds is still rarely reported. Summary of the invention

[0005] The object of the present invention is to provide an engineered Yarrowia lipolytica strain producing isoprenylated aromatic compounds, and a construction method and application thereof. Specifically, the present invention concurrently engineered the synthesis of two precursors, p-coumaric acid and dimethylallyl pyrophosphate (DMAPP), in two haploid Yarrowia lipolytica strains. The recipient strain was then mated with the donor strain to construct a diploid strain that efficiently synthesizes drupanin and artepillin C. Using a similar method, a strain producing 8-prenylnaringenin was also constructed. The present invention not only provides a basis for the sustainable production of drupanin, artepillin C and 8-prenylnaringenin, but also provides a new approach for rapidly engineering the yeast to produce other isoprenylated compounds. Based on the above research results, the present invention is completed.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides an engineered Yarrowia lipolytica strain that produces isopentenylated aromatic compounds. The engineered Yarrowia lipolytica strain is a diploid strain obtained by mating a DMAPP haploid donor strain with an aromatic compound haploid recipient strain; wherein the isopentenylated aromatic compounds include at least drupanin, artepillin C and 8-prenylnaringenin.

[0008] When the prenylated aromatic compounds are drupanin and artepillin C, the engineered Yarrowia lipolytica is obtained by mating a first DMAPP haploid donor bacterium with a p-coumaric acid haploid recipient bacterium.

[0009] The first DMAPP haploid donor bacteria is constructed by the following method: first, Yarrowia lipolytica overexpresses AtoB, HMGS, HMGR, ERG8, ERG19 and tAcPT1 genes, then inhibits the expression of ERG9, expresses heterologous MvaE, MvaS and MK genes, and replaces the MATA gene with the MATB gene.

[0010] Wherein, the starting strain first Yarrowia lipolytica can be the Yarrowia lipolytica strain Po1f;

[0011] The method of inhibiting the expression of ERG9 can be replacing the original promoter of ERG9 with the weak promoter F25773.

[0012] The p-coumaric acid haploid receptor bacteria are constructed by the following method: the second Yarrowia lipolytica expresses a heterologous TAL gene and overexpresses a gene mutant ARO4 that can relieve tyrosine feedback inhibition. K221L and ARO7 G139S Thus, the production of p-coumaric acid is increased. At the same time, the tAcPT1 gene is expressed to synthesize drupanin and artepillin C, and the original promoter of ERG9 is replaced by the weak promoter F25773, thereby inhibiting the expression of ERG9.

[0013] Wherein, the second Yarrowia lipolytica may be a Yarrowia lipolytica strain Po1fΔtrp;

[0014] The expressed heterologous TAL gene may be expressed in 1-3 copies of a heterologous TAL gene.

[0015] The method of inhibiting the expression of ERG9 can be replacing the original promoter of ERG9 with the weak promoter F25773.

[0016] When the prenylated aromatic compound is 8-prenylnaringenin, the engineered Yarrowia lipolytica is obtained by mating a second DMAPP haploid donor bacterium with a naringenin haploid recipient bacterium.

[0017] The second DMAPP haploid donor bacteria may be the first DMAPP haploid donor bacteria, or may be obtained by expressing a heterologous N8DT gene on the basis of the first DMAPP haploid donor bacteria.

[0018] The heterologous N8DT gene expressed may be a heterologous tN8DT gene obtained by expressing 1-2 copies of a truncated transit peptide.

[0019] The naringenin haploid recipient bacteria can express TAL-4CL, CHS and CHI for the second Yarrowia lipolytica, and overexpress YIARO4 K221L 、YlARO7 G139S and PEX10.

[0020] In another specific embodiment of the present invention, a Yarrowia lipolytica strain (Yarrowialipolytica) DD3 is provided, which has been deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province) on July 12, 2024, and its biological collection number is CCTCC NO: M 20241541.

[0021] In another specific embodiment of the present invention, a Yarrowia lipolytica strain (Yarrowialipolytica) DP4 is provided, which has been deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province) on July 12, 2024, and its biological collection number is CCTCC NO: M 20241542.

[0022] The second aspect of the present invention provides a method for constructing the above-mentioned Yarrowia lipolytica engineered bacteria that produces isopentenylated aromatic compounds, the construction method comprising the steps of mating a DMAPP haploid donor bacterium with an aromatic compound haploid recipient bacterium;

[0023] Wherein, the prenylated aromatic compounds at least include drupanin, artepillin C and 8-prenylnaringenin.

[0024] When the prenylated aromatic compounds produced by the Yarrowia lipolytica engineered bacteria are drupanin and artepillin C, the Yarrowia lipolytica engineered bacteria are obtained by mating a first DMAPP haploid donor bacterium with a p-coumaric acid haploid recipient bacterium.

[0025] The first DMAPP haploid donor bacteria is constructed by the following method: first, Yarrowia lipolytica overexpresses AtoB, HMGS, HMGR, ERG8, ERG19 and tAcPT1 genes, then inhibits the expression of ERG9, expresses heterologous MvaE, MvaS and MK genes, and replaces the MATA gene with the MATB gene.

[0026] Wherein, the starting strain first Yarrowia lipolytica can be the Yarrowia lipolytica strain Po1f;

[0027] The expression of ERG9 can be inhibited by replacing the original promoter of ERG9 with the weak promoter F25773.

[0028] The p-coumaric acid haploid receptor bacteria are constructed by the following method: the second Yarrowia lipolytica expresses a heterologous TAL gene and overexpresses a gene mutant ARO4 that can relieve tyrosine feedback inhibition. K221L and ARO7 G139S Thus, the production of p-coumaric acid is increased. At the same time, the tAcPT1 gene is expressed to synthesize drupanin and artepillin C, and the original promoter of ERG9 is replaced by the weak promoter F25773 to inhibit the expression of ERG9.

[0029] Wherein, the second Yarrowia lipolytica may be a Yarrowia lipolytica strain Po1fΔtrp;

[0030] The expressed heterologous TAL gene may be expressed in 1-3 copies of a heterologous TAL gene.

[0031] The method of inhibiting the expression of ERG9 can be replacing the original promoter of ERG9 with the weak promoter F25773.

[0032] When the prenylated aromatic compound produced by the Yarrowia lipolytica engineered bacteria is 8-prenylnaringenin, the Yarrowia lipolytica engineered bacteria is obtained by mating a second DMAPP haploid donor bacterium with a naringenin haploid recipient bacterium.

[0033] The second DMAPP haploid donor bacteria may be the first DMAPP haploid donor bacteria, or may be obtained by expressing a heterologous N8DT gene on the basis of the first DMAPP haploid donor bacteria.

[0034] Wherein, the heterologous N8DT gene expressed may be a heterologous tN8DT gene expressing 1-2 copies. Further, the heterologous N8DT gene may be a heterologous tN8DT gene obtained by truncating the transit peptide.

[0035] The naringenin haploid recipient bacteria can express TAL-4CL, CHS and CHI for the second Yarrowia lipolytica, and overexpress YIARO4 K221L 、YlARO7 G139S and PEX10.

[0036] The third aspect of the present invention provides the use of the above-mentioned Yarrowia lipolytica engineered bacteria in the production of prenylated aromatic compounds.

[0037] A fourth aspect of the present invention provides an industrial production method for isoprenoidized aromatic compounds, the industrial production method comprising: fermenting and culturing the above-mentioned Yarrowia lipolytica engineered bacteria; and separating and purifying to obtain the isoprenoidized aromatic compounds.

[0038] A fifth aspect of the present invention provides applications of the above-mentioned Yarrowia lipolytica engineered bacteria or the above-mentioned industrial production method in the fields of food, medicine and chemical industry.

[0039] Beneficial technical effects of one or more of the above technical solutions:

[0040] The above technical scheme discloses the synthesis of isoprenyl aromatic compounds using Yarrowia lipolytica and mating strategies. DMAPP and aromatic compounds are engineered in two haploid strains respectively. The aromatic compound strain is mated with the DMAPP strain to construct a diploid strain for synthesizing isoprenyl aromatic compounds, and finally an engineered Yarrowia lipolytica strain capable of high production of drupanin, artepillin C and 8-prenylnaringenin is successfully constructed, providing a new approach for the industrial production of isoprenyl compounds. Therefore, the above technical scheme has excellent practical application value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0042] Figure 1 The present invention utilizes Yarrowia lipolytica and a mating strategy to synthesize prenylated aromatic compounds. DMAPP and aromatic compounds are engineered in two haploid strains respectively. The aromatic compound strain is mated with the DMAPP strain to construct a diploid strain for synthesizing prenylated aromatic compounds.

[0043] Figure 2Schematic diagram of the present invention for the expression of isopentenyl transferase and engineering of mevalonate pathway in Yarrowia lipolytica to synthesize drupanin and artepillin C. (a) Schematic diagram of optimizing upstream pathway metabolic flux to increase DMAPP supply. (b) Yields of drupanin and artepillin C produced by M1, M6 and M10 strains.

[0044] Figure 3 The present invention improves the availability of p-coumaric acid as an acceptor substrate. (a) Schematic diagram of the optimization of the p-coumaric acid biosynthetic pathway. (b) The yield of p-coumaric acid under different engineering strategies. (c) The yield of drupanin and artepillin C in p-coumaric acid biosynthetic strains.

[0045] Figure 4 The present invention synthesizes drupanin and artepillin C de novo by mating donor and recipient strains. (a) Verify the genotype of the diploid strain using nutrient-deficient medium. (b) The yield of drupanin and artepillin C in the diploid strain.

[0046] Figure 5 The production of drupanin and artepillin C in the fed-batch culture of the present invention. (a) The production of drupanin and artepillin C by the DD3 strain in a 3-L bioreactor by fed-batch fermentation. (b) The OD600 and p-coumaric acid production of the strain in the fed-batch fermentation.

[0047] Figure 6 The present invention is a construction of a strain producing the receptor substrate naringenin and the de novo synthesis of 8-prenylnaringenin after mating. (a) An overview of the biosynthetic pathway for synthesizing 8-prenylnaringenin from glucose. TAL encodes tyrosine ammonia lyase from Flavobacterium johnsoniae, 4CL encodes 4-coumarate-CoA ligase from Arabidopsis thaliana, CHS encodes chalcone synthase from Hypericum androsaemum, and CHI encodes chalcone isomerase from Medicago sativa. (b) The introduction and optimization of the naringenin biosynthetic pathway increased the yield of naringenin. (c) The yield of 8-prenylnaringenin in strains expressing different copies of the tN8DT gene. (d) The yield of 8-prenylnaringenin in a diploid strain. (e) The yield and OD600 value of 8-prenylnaringenin and naringenin of the DP4 strain in fed-batch fermentation. DETAILED DESCRIPTION

[0048] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0049] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0050] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels.

[0051] Example

[0052] 1 Materials and Methods

[0053] 1.1 Strains, culture media and culture conditions

[0054] E. coli was cultured at 37°C using Luria-Bertani medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl) containing 100 mg / mL ampicillin. Yarrowia lipolytica was cultured in YPD medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) or (SC) defective medium (20 g / L glucose, 1.7 g / L yeast nitrogen base, 5 g / L ammonium sulfate and CSM). If necessary, antibiotics such as hygromycin B (500 mg / L), bleomycin (800 mg / L) or nourseothricin (100 mg / L) were added to YPD and SC medium. Shake flask fermentation was carried out in a 300 mL flask with 50 mL of YPD medium and cultured at 30°C with shaking at 200 rpm. YMC medium (5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract, 0.5 g / L sodium citrate).

[0055] 1.2 Plasmid construction and genetic engineering

[0056] E. coli DH5α was used for plasmid construction. Heterologous genes were codon optimized and synthesized by Genscript (Nanjing, China). Plasmids were constructed by Gibson assembly. Target DNA fragments were obtained by polymerase chain reaction (PCR) using Phanta Max ultra-high fidelity DNA polymerase (Vazyme, Nanjing, China). Restriction endonucleases were purchased from Thermo Fisher Scientific (Shanghai, China). The strains and plasmids used in this study are shown in Tables 1-2 below. Genes are shown in SEQ ID NOs. 1-14.

[0057] Heterologous and endogenous genes are expressed by constructing them into plasmids under the control of strong constitutive promoters, and then linearizing the plasmids and transforming them for random integration into the genome. Gene deletion is performed by transforming a plasmid containing Cas9 and sgRNA targeting the corresponding gene, and transformants with frameshift mutations are selected as gene deletion strains. Site-specific integration requires the simultaneous transformation of two plasmids, one containing sgRNA and Cas9, and the other containing the DNA to be integrated, with the upstream and downstream of the DNA being the homology arms of the integration site.

[0058] Table 1 The strains selected in this example

[0059]

[0060]

[0061] Table 2 This embodiment

[0062]

[0063]

[0064]

[0065] 1.3 Product extraction and quantification

[0066] For the detection of p-coumaric acid and naringenin, the fermentation supernatant was mixed with an equal volume of methanol and vortexed thoroughly. After centrifugation, the supernatant was filtered and analyzed by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) using a C18 column (4.6 × 250 mm, Shimadzu) at 30 °C. For naringenin analysis, the mobile phase consisted of acetonitrile and water. For p-coumaric acid detection, the mobile phase consisted of acetonitrile and 10 mM ammonium formate solution.

[0067] To detect drupanin and artepillin C, the fermentation broth was mixed with an equal volume of ethyl acetate and quartz sand. The cells were disrupted by a high-throughput tissue grinding and disruption instrument (Shangkezhi, China). After centrifugation, the organic phase was collected, dried with nitrogen, and dissolved in methanol. After the sample was filtered, it was analyzed by ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer (SCIEXTriple Quad TM 5500+) was used for analysis. Chromatographic separation was performed using a Phenomenex C18 column (100×2.1 mm, particle size 2.6 μm) at a column temperature of 35°C. The mobile phase consisted of acetonitrile and water.

[0068] 1.4 Mating method

[0069] Haploid strains were cultured in YPD medium at 30°C for 12 hours. Subsequently, cells were washed twice with mating medium. For mating, an equal volume of strains of opposite mating type was resuspended in mating medium and cultured at 28°C for 24 hours. After mating, cells were plated on SC-URA-TRP selective medium to select diploid strains.

[0070] 1.5 Spot test

[0071] After overnight culture, cells were washed twice with sterile water and OD600 was adjusted to 1. Cell density was diluted with sterile water from 10^-1 to 10^-4. 4 μL of the dilution was spotted onto SC-URA, SC-TRP and SC-URA-TRP plates, and the plates were cultured at 30°C for 3 days.

[0072] 1.6 Fed-batch fermentation in bioreactors

[0073] The strain was inoculated into a test tube containing YPD medium as a primary seed culture, and cultured at 30°C with 220rpm shaking for 24 hours. 20% of the culture solution was inoculated into a 1-L shake flask containing 180mL YPD medium, and cultured at 30°C with 220rpm shaking. Then, 10% of the secondary culture solution was inoculated into a 3-L bioreactor (FS-02-D03P; Winpact, Saratoga, CA, USA) to start fermentation. The initial culture medium included 100g / L glucose, 40g / L peptone and 20g / L yeast extract. The optimal conditions for fed-batch fermentation were 30°C, 900rpm, pH 5.5 and 2v / v / m. Before 144 hours, when the glucose concentration was lower than 2g / L, a solution of 600g / L glucose, 40g / L peptone and 20g / L yeast extract was supplemented. Between 144 and 240 hours, when the glucose concentration was lower than 2 g / L, 600 g / L glucose was added. Artepillin C, drupanin and biomass in the fermentation broth were also measured.

[0074] result

[0075] 2.1 Biosynthesis of Drupanin and Artepillin C

[0076] The aromatic compound acceptor p-coumaric acid and the isopentenyl donor DMAPP can biosynthesize drupanin and artepillin C under the catalysis of prenyltransferase AcPT1. Figure 2 a). In the Po1f strain, AtoB from Escherichia coli, HMGS endogenous to Yarrowia lipolytica, HMGR from Bordetella petrii, ERG8 and ERG19 endogenous to Yarrowia lipolytica were expressed, Ku70 was knocked out, and tAcPT1 was site-directedly integrated at the B3 site to form the M1 strain. When 500 mg / L p-coumaric acid was added, the M1 strain produced 0.62 mg / L of drupanin and 0.01 mg / L of artepillin C ( Figure 2 b). We used the F25773 promoter to control the expression of ERG9 to obtain the M6 ​​strain, and the production of drupanin and artepillin C reached 2.19 mg / L and 0.13 mg / L, respectively ( Figure 2 b). We then expressed MvaE and MvaS from Enterococcus faecalis and MK from Methanosarcina mazei in the M6 ​​strain, and the production of drupanin and artepillin C was further increased to 3.54 mg / L and 0.79 mg / L, respectively ( Figure 2 b).

[0077] 2.2 Construction of p-coumaric acid receptor strain

[0078] At the same time, we constructed a p-coumaric acid producing strain ( Figure 3 a). We expressed the TAL gene from Flavobacterium johnsoniae in Po1fΔtrp, and the resulting Cou1 was able to produce 205 mg / L of p-coumaric acid ( Figure 3 b). We then integrated two additional copies of the TAL gene, and the production of p-coumaric acid increased to 589 mg / L, a 1.8-fold increase over Cou1 ( Figure 3 b) We then overexpressed the feedback inhibition resistance gene ARO4 K221L and ARO7 G139S , further increasing the production of p-coumaric acid to 789 mg / L, forming the Cou3 strain ( Figure 3 b). We expressed tAcPT1 in Cou3 and replaced the ERG9 promoter with the F25773 promoter to obtain the Cou7 strain, which produced drupanin and artepillin C of 0.37 mg / L and 0.0005 mg / L, respectively ( Figure 3 c).

[0079] 2.3 De novo synthesis of drupanin and artepillin C by yeast mating

[0080] We replaced the matA1 and matA2 genes in M10 with matB1 and matB2 genes to form M11. M11 was then mated with the Cou7 strain. The strain obtained after mating was verified for ploidy using SC-URA-TRP, because the haploid strains M11 (uracil-deficient, tryptophan prototrophy) and Cou7 (uracil-prototrophy, tryptophan-deficient) could not grow on SC-URA-TRP plates ( Figure 4 a). We selected six diploid strains to test the production of drupanin and artepillin C. In the absence of p-coumaric acid, the production of drupanin and artepillin C ranged from 0.64–1.06 mg / L and 0.01–0.03 mg / L, respectively ( Figure 4 b).

[0081] 2.4 Fed-batch fermentation for production of drupanin and artepillin C

[0082] Fed-batch fermentation of strain DD3 in a 3-L bioreactor yielded 52.26 mg / L drupanin and 7.45 mg / L artepillin C, respectively. Figure 5 a). At the same time, the strain produced 919 mg / L of p-coumaric acid ( Figure 5 b).

[0083] 2.5 De ​​novo synthesis of 8-prenylnaringenin using a mating strategy

[0084] We then attempted to synthesize 8-prenylnaringenin ( Figure 6 a). Our strategy to construct the naringenin biosynthesis pathway in Yarrowia lipolytica was to simultaneously integrate TAL-4CL, CHS, and CHI into the genome of the Po1fΔtrp strain, and the naringenin production of the Nar4 strain reached 48 mg / L ( Figure 6 b). Subsequently, we overexpressed PEX10 (encoding peroxisomal matrix protein) ( Figure 6 a). Naringenin production reached 77mg / L ( Figure 6 b) Overexpression of YIARO4 K221L and YlARO7 G139S The feedback inhibition resistance mutation increased the production of naringenin by 60% to 77 mg / L ( Figure 6 b) We used YARO4 from Yarrowia lipolytica K221L and YlARO7 G139S The Nar12 strain was constructed by co-expression with PEX10 ( Figure 6 b). The strain produced 91 mg / L naringenin.

[0085] We expressed the prenyltransferase N8DT with a truncated transit peptide, and the yield of 8-prenylnaringenin was 0.18 mg / L ( Figure 6 c). The production of 8-prenylnaringenin in the strain with two copies of tN8DT gene increased to 0.69 mg / L ( Figure 6 c). We then mated PN5 with the isopentenyl donor strain M11. The production of 8-prenylnaringenin was approximately 0.27-0.57 mg / L ( Figure 6 d), the production of naringenin was about 114-184 mg / L. The results of fed-batch fermentation of DP4 strain showed that the production of 8-prenylnaringenin reached 4.36 mg / L ( Figure 6 e). In fed-batch fermentation, the strain produced 444 mg / L of naringenin, OD 600 Reached 194 ( Figure 6 e).

[0086] Related gene information used in the present invention:

[0087]

[0088] The nucleotide sequence information of the gene codon optimized used in the present invention is:

[0089]

[0090] References

[0091] Cui,Z.,Jiang,X.,Zheng,H.,Qi,Q.,Hou,J.,2019.Homology-independentgenome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica.Biotechnology and bioengineering.116,354-363.

[0092] Gao,S.,Tong,Y.,Wen,Z.,Zhu,L.,Ge,M.,Chen,D.,Jiang,Y.,Yang,S.,2016.Multiplex gene editing of the Yarrowia lipolytica genome using theCRISPR-Cas9 system.Journal of industrial microbiology&biotechnology.43,1085-93.

[0093] Liu, X., Cui, Z., Su, T., Lu,

[0094] Liu,Y.,Jiang,X.,Cui,Z.,Wang,Z.,Qi,Q.,Hou,J.,2019.Engineering theoleaginous yeast Yarrowia lipolytica for production ofα-farnesene.Biotechnology for biofuels.12,296.

[0095] Liu, Y., Zhang, J., Li, Q., Wang, Z., Cui, Z., Su, T., Lu,

[0096] Zhu,Y.,Liu,J.,Sun,L.,Liu,M.,Qi,Q.,Hou,J.,2024.Development of geneticmarkers in Yarrowia lipolytica.Applied Microbiology and Biotechnology.108,14.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered strain of Yarrowia lipolytica producing prenylated aromatic compounds, characterized in that: The Yarrowia lipolytica engineered bacteria is a diploid strain obtained by mating a DMAPP haploid donor bacterium with an aromatic compound haploid recipient bacterium; wherein the isoprenylated aromatic compound includes at least drupanin, artepillin C and 8-prenylnaringenin.

2. The engineered Yarrowia lipolytica according to claim 1, characterized in that When the prenylated aromatic compounds are drupanin and artepillin C, the engineered Yarrowia lipolytica is obtained by mating a first DMAPP haploid donor bacterium with a p-coumaric acid haploid recipient bacterium.

3. The engineered Yarrowia lipolytica according to claim 2, characterized in that The first DMAPP haploid donor bacteria is constructed by the following method: first, Yarrowia lipolytica overexpresses AtoB, HMGS, HMGR, ERG8, ERG19 and tAcPT1 genes, then inhibits the expression of ERG9, expresses heterologous MvaE, MvaS and MK genes, and replaces the MATA gene with the MATB gene; Further, the first starting strain Yarrowia lipolytica is the Yarrowia lipolytica strain Po1f; The method of inhibiting the expression of ERG9 is to replace the original promoter of ERG9 with the weak promoter F25773.

4. The engineered Yarrowia lipolytica according to claim 2, characterized in that The p-coumaric acid haploid receptor bacteria are constructed by the following method: the second Yarrowia lipolytica expresses a heterologous TAL gene and overexpresses the feedback inhibition resistance gene ARO4 K221L 、ARO7 G139S and tAcPT1 genes, inhibiting the expression of ERG9.

5. The engineered Yarrowia lipolytica according to claim 2, characterized in that The second Yarrowia lipolytica is the Yarrowia lipolytica strain Po1fΔtrp; The expression of heterologous TAL gene is the expression of 1-3 copies of heterologous TAL gene; The method of inhibiting the expression of ERG9 is to replace the original promoter of ERG9 with the weak promoter F25773.

6. The engineered Yarrowia lipolytica according to claim 1, characterized in that When the prenylated aromatic compound is 8-prenylnaringenin, the Yarrowia lipolytica engineered bacterium is obtained by mating a second DMAPP haploid donor bacterium with a naringenin haploid recipient bacterium; Wherein, the second DMAPP haploid donor bacteria is the first DMAPP haploid donor bacteria, or is obtained by expressing a heterologous N8DT gene on the basis of the first DMAPP haploid donor bacteria; The heterologous N8DT gene expressed is a heterologous N8DT gene expressing 1-2 copies; further, the heterologous N8DT gene is a heterologous N8DT gene obtained by truncating the transit peptide; The naringenin haploid recipient bacteria is the second Yarrowia lipolytica expressing TAL-4CL, CHS and CHI, and overexpressing YIARO4 K221L 、YlARO7 G139S and PEX10.

7. The engineered Yarrowia lipolytica according to claim 1, characterized in that The engineered Yarrowia lipolytica is a Yarrowia lipolytica strain (Yarrowia lipolytica) DD3, which has been deposited in the China Center for Type Culture Collection on July 12, 2024, and its biological preservation number is CCTCC NO: M 20241541; or, the engineered Yarrowia lipolytica is a Yarrowia lipolytica strain (Yarrowia lipolytica) DP4, which has been deposited in the China Center for Type Culture Collection on July 12, 2024, and its biological preservation number is CCTCC NO: M 20241542.

8. The method for constructing an engineered strain of Yarrowia lipolytica producing isopentenylated aromatic compounds according to any one of claims 1 to 7, characterized in that: The construction method comprises the steps of mating a DMAPP haploid donor bacterium and an aromatic compound haploid recipient bacterium; Wherein, the isopentenylated aromatic compounds at least include drupanin, artepillin C and 8-prenylnaringenin.

9. An industrial production method of isoprenated aromatic compounds, characterized in that: The industrial production method comprises: fermenting and culturing the Yarrowia lipolytica engineered bacteria according to any one of claims 1 to 7; and separating and purifying to obtain the prenylated aromatic compounds.

10. Use of the engineered Yarrowia lipolytica according to any one of claims 1 to 7 or the industrial production method according to claim 9 in the fields of food, medicine and chemical industry.

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